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Inflammation is a physiological state where immune cells evoke a response against detrimental insults. Finding a safe and effective treatment for inflammation associated diseases has been a challenge. In this regard, human mesenchymal stem cells (hMSC), exert immunomodulatory effects and have regenerative capacity making it a promising therapeutic option for resolution of acute and chronic inflammation. T cells play a critical role in inflammation and depending on their phenotype, they can stimulate or suppress inflammatory responses. However, the regulatory effects of hMSC on T cells and the underlying mechanisms are not fully elucidated. Most studies focused on activation, proliferation, and differentiation of T cells. Here, we further investigated memory formation and responsiveness of CD4 + T cells and their dynamics by immune-profiling and cytokine secretion analysis. Umbilical cord mesenchymal stem cells (UC-MSC) were co-cultured with either αCD3/CD28 beads, activated peripheral blood mononuclear cells (PBMC) or magnetically sorted CD4 + T cells. The mechanism of immune modulation of UC-MSC were investigated by comparing different modes of action; transwell, direct cell-cell contact, addition of UC-MSC conditioned medium or blockade of paracrine factor production by UC-MSC. We observed a differential effect of UC-MSC on CD4 + T cell activation and proliferation using PBMC or purified CD4 + T cell co-cultures. UC-MSC skewed the effector memory T cells into a central memory phenotype in both co-culture conditions. This effect on central memory formation was reversible, since UC-MSC primed central memory cells were still responsive after a second encounter with the same stimuli. The presence of both cell-cell contact and paracrine factors were necessary for the most pronounced immunomodulatory effect of UC-MSC on T cells. We found suggestive evidence for a partial role of IL-6 and TGFβ in the UC-MSC derived immunomodulatory function. Collectively, our data show that UC-MSCs clearly affect T cell activation, proliferation and maturation, depending on co-culture conditions for which both cell-cell contact and paracrine factors are needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sengun, Wolfs, van Bruggen, van Cranenbroek, Simonetti, Ophelders, de Jonge, Joosten and van der Molen.)

Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/β receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4 + T-cell compartment. Here, we show low IFN-β-mediated inhibition of CD4 + T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4 + T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-β in contrast to adults. The distinct IFN-β-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jans, Unger, Raeven, Simonetti, Eleveld, de Groot, de Jonge and Ferwerda.)

Background: Respiratory syncytial virus (RSV) is a common cause of bronchiolitis in infants with a wide spectrum of disease severity. Besides environmental and genetic factors, it is thought that the innate immune system plays a pivotal role. The aim of this study was to investigate the expression of immune receptors on monocytes and the in vitro responsiveness from infants with severe RSV infections., Methods: Peripheral blood mononuclear cells (PBMCs) from infants with RSV infections were isolated. Classical, intermediate and nonclassical monocytes were immunophenotyped for the expression of CD14, CD16, human leukocyte antigen (HLA)-ABC and HLA-DR. PBMCs were stimulated with lipopolysaccharide to determine the secretion of tumor necrosis factor and interleukin (IL)-10 with enzyme-linked immunosorbent assay., Results: During RSV infection, intermediate monocytes are increased in the peripheral blood, whereas classical and nonclassical monocytes are reduced. The expression of CD14 and HLA-ABC is increased on monocytes, whereas the expression of HLA-DR is suppressed. Low HLA-DR expression is correlated with increased disease severity. PBMCs from infants with severe RSV infections show an impaired IL-10 response in vitro., Conclusions: Phenotyping subpopulations of monocytes combined with in vitro responsiveness reveals significant differences between nonsevere and severe RSV infections. Reduced HLA-DR expression and impaired IL-10 production in vitro during severe RSV infections indicate that an imbalanced innate immune response may play an important role in disease severity.

Context: Participation in proficiency testing (PT) or external quality assessment (EQA) programs allows the assessment and comparison of test performance among different clinical laboratories and technologies. In addition to the approximately 2300 tests for individual genetic disorders, recent advances in technology have enabled the development of clinical tests that quickly and economically analyze the entire human genome. New PT/EQA approaches are needed to ensure the continued quality of these complex tests., Objectives: To review the availability and scope of PT/EQA for molecular genetic testing for inherited conditions in Europe, Australasia, and the United States; to evaluate the successes and demonstrated value of available PT/EQA programs; and to examine the challenges to the provision of comprehensive PT/EQA posed by new laboratory practices and methodologies., Data Sources: The available literature on this topic was reviewed and supplemented with personal experiences of several PT/EQA providers., Conclusions: Proficiency testing/EQA schemes are available for common genetic disorders tested in many clinical laboratories but are not available for most genetic tests offered by only one or a few laboratories. Provision of broad, method-based PT schemes, such as DNA sequencing, would allow assessment of many tests for which formal PT is not currently available. Participation in PT/EQA improves the quality of testing by identifying inaccuracies that laboratories can trace to errors in their testing processes. Areas of research and development to ensure that PT/EQA programs can meet the needs of new and evolving genetic tests and technologies are identified and discussed.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias resulting in short-limbed dwarfism, joint pain, and stiffness. PSACH and the largest proportion of autosomal dominant MED (AD-MED) results from mutations in cartilage oligomeric matrix protein (COMP); however, AD-MED is genetically heterogenous and can also result from mutations in matrilin-3 (MATN3) and type IX collagen (COL9A1, COL9A2, and COL9A3). In contrast, autosomal recessive MED (rMED) appears to result exclusively from mutations in sulphate transporter solute carrier family 26 (SLC26A2). The diagnosis of PSACH and MED can be difficult for the nonexpert due to various complications and similarities with other related diseases and often mutation analysis is requested to either confirm or exclude the diagnosis. Since 2003, the European Skeletal Dysplasia Network (ESDN) has used an on-line review system to efficiently diagnose cases referred to the network prior to mutation analysis. In this study, we present the molecular findings in 130 patients referred to ESDN, which includes the identification of novel and recurrent mutations in over 100 patients. Furthermore, this study provides the first indication of the relative contribution of each gene and confirms that they account for the majority of PSACH and MED., (© 2011 Wiley Periodicals, Inc.)

Prader Willi and Angelman syndromes are clinically distinct genetic disorders both mapping to chromosome region 15q11-q13, which are caused by a loss of function of paternally or maternally inherited genes in the region, respectively. With clinical diagnosis often being difficult, particularly in infancy, confirmatory genetic diagnosis is essential to enable clinical intervention. However, the latter is challenged by the complex genetics behind both disorders and the unmet need for characterised reference materials to aid accurate molecular diagnosis. With this in mind, a panel of six genotyping reference materials for Prader Willi and Angelman syndromes was developed, which should be stable for many years and available to all diagnostic laboratories. The panel comprises three Prader Willi syndrome materials (two with different paternal deletions, and one with maternal uniparental disomy (UPD)) and three Angelman syndrome materials (one with a maternal deletion, one with paternal UPD or an epigenetic imprinting centre defect, and one with a UBE3A point mutation). Genomic DNA was bulk-extracted from Epstein-Barr virus-transformed lymphoblastoid cell lines established from consenting patients, and freeze-dried as aliquots in glass ampoules. In total, 37 laboratories from 26 countries participated in a collaborative study to assess the suitability of the panel. Participants evaluated the blinded, triplicate materials using their routine diagnostic methods against in-house controls or externally sourced uncertified reference materials. The panel was established by the Expert Committee on Biological Standardization of the World Health Organization as the first International Genetic Reference Panel for Prader Willi and Angelman syndromes.

Aim: To conduct a pragmatic, randomized controlled trial to assess whether thiopurine methyltransferase (TPMT) genotyping prior to azathioprine reduces adverse drug reactions (ADRs)., Methods: A total of 333 participants were randomized 1:1 to undergo TPMT genotyping prior to azathioprine or to commence treatment without genotyping., Results: There was no difference in the primary outcome of stopping azathioprine due to an adverse reaction (ADR, p = 0.59) between the two study arms. ADRs were more common in older patients (p = 0.01). There was no increase in stopping azathioprine due to ADRs in TPMT heterozygotes compared with wild-type individuals. The single individual with TPMT variant homozygosity experienced severe neutropenia., Conclusion: Our work supports the strong evidence that individuals with TPMT variant homozygosity are at high risk of severe neutropenia, whereas TPMT heterozygotes are not at increased risk of ADRs at standard doses of azathioprine.

Antibody-mediated killing of Streptococcus pneumoniae (pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies by use of a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and required for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis-of-variance (ANOVA) mixed-effects model, provided a common reference for assessing agreement among these laboratories. Agreement was evaluated in terms of assay accuracy, reproducibility, repeatability, precision, and bias. We also reviewed four acceptance criterion intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy, and concordance results among laboratories and the consensus titers revealed acceptable agreement. The results of this study indicate that the bioassays evaluated in this study are robust, and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template for evaluating future multilaboratory studies.

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by expansion of a trinucleotide (CGG)n repeat sequence in the 5' untranslated region of the FMR1 gene, resulting in promoter hypermethylation and suppression of FMR1 transcription. Additionally, pre-mutation alleles in carrier males and females may result in Fragile X tremor ataxia syndrome and primary ovarian insufficiency, respectively. Fragile X is one of the most commonly requested molecular genetic tests worldwide. Quality assessment schemes have identified a wide disparity in allele sizing between laboratories. It is therefore important that clinical laboratories have access to characterized reference materials (RMs) to aid accurate allele sizing and diagnosis. With this in mind, a panel of genotyping RMs for Fragile X syndrome has been developed, which should be stable over many years and available to all diagnostic laboratories. Immortalized cell lines were produced by Epstein-Barr virus transformation of lymphocytes from consenting patients. Genomic DNA was extracted in bulk and RM aliquots were freeze-dried in glass ampoules. Twenty-one laboratories from seventeen countries participated in a collaborative study to assess their suitability. Participants evaluated the samples (blinded, in triplicate) in their routine methods alongside in-house and commercial controls. The panel of five genomic DNA samples was endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the World Health Organization.

Background: Lipid-induced modulation of phagocyte function seems to contribute to increased susceptibility to infections in patients on parenteral nutrition, and an increased risk for development of pneumonia has been observed in this group. The role of various structurally different lipid emulsions, however, remains unclear. In this study, we therefore assessed phagocyte function, as the capacity of neutrophils to eliminate Streptococcus pneumoniae (i.e. combined result of phagocytosis and killing), in the presence of these lipids., Materials and Methods: Neutrophils from six healthy volunteers were incubated for 1 h in emulsions (5 mmol L(-1)) derived from soybean- (LCT), fish- (VLCT), olive- (LCT-MUFA), mixed soybean/coconut oils (LCT/MCTs) or structured lipids (SL). After opsonization of the pneumococci (strain OREP-4) by human immunoglobulins, bacteria and neutrophils were incubated in the presence of complement. Next, pneumococcal elimination was evaluated and expressed as the percentage of bacteria eliminated relative to the initial bacterial numbers in neutrophil-free samples., Results: Neutrophils that were not exposed to lipids showed a pneumococcal elimination capacity of 75 +/- 3% (mean +/- SD). This significantly decreased after exposure to LCT-MUFA (70 +/- 6%), VLCT (67 +/- 2%), SL (63 +/- 9%), LCT (66 +/- 10%) and LCT/MCT (47 +/- 15%)., Conclusion: These data demonstrate that parenteral lipids impair the microbial elimination capacity of neutrophils in a structure-dependent manner. In accordance with our previously reported in vitro effect on a range of phagocyte functions, LCT/MCT is by far the most potent in this respect.

The European Molecular Genetics Quality Network (EMQN) has been organizing an external quality assessment (EQA) scheme for molecular genetic testing of trinucleotide repeat mutations in the spinocerebellar ataxias (SCAs) since 2004. DNA samples were validated by at least two independent labs and two different methods. Together with mock clinical case descriptions and requests for specific SCA gene analyses, these were sent to registered participants each year. Laboratories were asked to use their routine procedures and protocols. A panel of assessors reviewed the final returns, including genotype results and reports, to assess the quality of (1) genotyping and (2) interpretation and reporting. A description of methods and raw data were also requested and were very useful for the final analysis. Altogether, during 3 years, 239 reports were received from the laboratories. Overall genotype error rate ranged 1.1-5.2%, a significant cause of concern. Scores for interpretation and reporting also showed that there is still much room for progress, although performance has improved over this period of assessment. The consequences of suboptimal laboratory practices, genotyping errors and misdiagnosis and of incorrect or incomplete interpretation and reporting have wide implications for patient lives, as well as for health management and counselling of relatives. EQA schemes are an important part of quality assurance in molecular genetic laboratories, and their use should become a routine part of laboratory diagnostic practice. Current evidence shows also that it is important that laboratories participate on a yearly basis and that this becomes mandatory for reference laboratories.

Objective: To compare data on the practices of molecular genetic testing (MGT) in laboratories in the United States with those in 18 other countries., Methods: A Web-based survey of MGT laboratory directors (n = 827; response rate 63%) in 18 countries on three continents was carried out, and the response from U.S. laboratories compared to all others. Quality assurance and reporting indices were developed and calculated for each responding laboratory., Results: A comparison of U.S. results with all other countries identified differences in laboratory setting, personnel qualifications, and the specific tests being offered, but similar rates of adherence to MGT quality standards and reporting practices were found. The survey also documented substantial transborder flow of specimens, most commonly due to the lack of availability of the test in the United States or because the test was available only through a research protocol, highlighting the need for common reporting and practice guidelines for the international MGT community., Conclusion: The findings presented here provide further support for the need to consider the application of the Organisation for Economic Cooperation and Development (OECD) Guidelines and the establishment of compatible accreditation programs or equivalent mechanisms across national borders to ensure the quality of laboratory services and the clinical usefulness of molecular genetic test reports for referred specimens.

Objectives: To validate the use of Real Time PCR, a widely used technique that can detect very low levels of Y chromosomal sequence, and to assess the use of a highly sensitive PCR technique, pyrophosphorolysis-activated polymerisation (PAP), for fetal sex determination using free fetal DNA (ffDNA)., Methods: The fetal sex was determined by Real Time PCR in 58 pregnancies using ffDNA isolated from maternal plasma. In parallel with the Real Time PCR experiments, the presence of Y chromosome sequence was also determined using PAP on 54 isolated ffDNA samples., Results: Both techniques detected Y chromosome sequence at very low levels with 98% specificity and 100% sensitivity (Real Time n = 44, PAP n = 54). Furthermore, the PAP technique was shown to be more robust than the Real Time PCR as none of the samples tested failed to meet the acceptance criteria. Combining the two techniques for male fetal sex detection from maternal blood plasma increases the sensitivity and specificity to 100% in this series., Conclusions: This study shows that both Real Time PCR and PAP can be used for Y chromosome detection on ffDNA. Furthermore, by using PAP in combination with Real Time PCR more reliable early prenatal sexing can be performed using ffDNA.

Skeletal dysplasias are difficult to diagnose for the nonexpert. In a previous study of patients with multiple epiphyseal dysplasia (MED), we identified cartilage oligomeric matrix protein (COMP) mutations in only 36% of cases and suspected that the low-mutation detection rate was partially due to misdiagnosis. We therefore instituted a clinical-radiographic review system, whereby all cases were evaluated by a panel of skeletal dysplasia experts (European Skeletal Dysplasia Network). Only those patients in whom the diagnosis of MED was confirmed by the panel were screened for mutations. Under this regimen the mutation detection rate increased to 81%. When clinical-radiological diagnostic criteria were relaxed the mutation rate dropped to 67%. We conclude that expert clinical-radiological review can significantly enhance mutation detection rates and should be part of any diagnostic mutation screening protocol for skeletal dysplasias.

Objective: To collect data on the practices of molecular genetic testing (MGT) laboratories for the development of national and international policies for quality assurance (QA)., Methods: A web-based survey of MGT laboratory directors (n = 827; response rate 63%) in 18 countries on 3 continents. QA and reporting indices were developed and calculated for each responding laboratory., Results: Laboratory setting varied among and within countries, as did qualifications of the directors. Respondents in every country indicated that their laboratory receives specimens from outside their national borders (64%, n = 529). Pair-wise comparisons of the QA index revealed a significant association with the director having formal training in molecular genetics (p < 0.005), affiliation with a genetics unit (p = 0.003), accreditation of the laboratory (p < 0.005) and participation in proficiency testing (p < 0.005). Research labs had a lower mean report score compared to all other settings (p < 0.05) as did laboratories accessioning <150 samples per year., Conclusion: MGT is provided under widely varying conditions and regulatory frameworks. The data provided here may be a useful guide for policy action at both governmental and professional levels., (Copyright 2007 S. Karger AG, Basel.)

Objective: Recent clinical trials of adoptive immunotherapy showed diminished reactivity of human T cells upon ex vivo manipulation. For a safe and effective clinical application of human T cells, it is necessary to improve ex vivo manipulation procedures and evaluate their impact on in vivo functionality. However, there is no preclinical model for quantitative assessment of in vivo functionality of human T cells. In this study, we investigated the feasibility of using the huPBMC- RAG2(-/-)gammac(-/-) xenogeneic mouse model. As a first example, we compared 3 different ex vivo culture conditions for human T cells., Methods: RAG2(-/-)gammac(-/-) mice received cultured human T cells that were stimulated via CD3 alone or costimulated via CD28 (CD3/28) and/or human 4-1BB (CD3/28/4-1BB). Engraftment levels and survival of the cells were measured. The dynamics of the human T cell phenotypes were analyzed during culture and in vivo, as well as the mechanism of the xenoresponse., Results: Engraftment potential was improved twofold for costimulation compared to CD3 alone (p < 0.001). Phenotypic analysis showed a strikingly similar pattern of development towards CD4(+) and CD8(+) effector and effector-memory cells, suggesting antigen-driven survival and expansion. All parameters used to analyze different effects on in vivo T-cell functionality, like culture condition, engraftment levels, survival of the cells over time, or xenogeneic graft-vs-host disease were absolutely independent of the distribution of the T cell population in vivo following contact with xeno-antigen., Conclusion: The huPBMC-RAG2(-/-)gammac(-/-) xenogeneic transplant model is the most sensitive to date for in vivo functional evaluation of human T cells.

Purpose: Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4+CD25+ regulatory T cells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell-dependent xenogeneic (x)-GvHD in a new model where x-GvHD is induced in RAG2-/-gammac-/- mice by i.v. administration of human peripheral blood mononuclear cells (PBMC)., Experimental Design: Human PBMC, depleted of or supplemented with autologous CD25+ Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of human T cells, and secretion of human cytokines were monitored at weekly intervals., Results: Depletion of CD25+ cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25+ cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4+ and CD8+ T cells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-gamma, suggesting the de novo development of TR1 cells., Conclusions: These results show, for the first time, the potent in vivo capacity of naturally occurring human Tregs to control GvHD-inducing autologous T cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring human Tregs.

Background: In the past 15 years, clinical laboratory science has been transformed by the use of technologies that cross the traditional boundaries between laboratory disciplines. However, during this period, issues of quality have not always been given adequate attention. The European Molecular Genetics Quality Network (EMQN) has developed a novel external quality assessment scheme for evaluation of DNA sequencing. We report the results of an international survey of the quality of DNA sequencing among 64 laboratories from 21 countries., Methods: Current practice for DNA sequence analysis was established by use of an online questionnaire. Participating laboratories were provided with 4 DNA samples of validated genotype. Evaluation of the results included assessing the quality of sequence data, variant genotypes, and mutation nomenclature. To accommodate variations in mutation nomenclature, variants indicated by participants were scored for compliance with 3 acceptable marking schemes., Results: A total of 346 genotypes were analyzed. Of these, 19 (5%) genotyping errors were made. Of these, 10 (53%) were false-negative and 9 (47%) were false-positive results. A further 27 (8%) errors were made in naming mutations. Results were analyzed for 3 indicators of data quality: PHRED quality scores, Quality Read Length, and Quality Read Overlap. Most laboratories produced results of acceptable diagnostic quality as judged by these indicators. The results were used to calculate a consensus benchmark for DNA sequencing against which individual laboratories could rank their performance., Conclusions: We propose that the consensus benchmark can be used as a baseline against which the aggregate and individual laboratory standard of DNA sequencing may be tracked from year to year.

The neural cell adhesion molecule L1 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs). Its expression is essential during embryonic development of the nervous system and it is involved in cognitive function and memory. Mutations in the L1CAM gene are responsible for four related L1 disorders; X-linked hydrocephalus/HSAS (Hydrocephalus as a result of Stenosis of the Aqueduct of Sylvius), MASA (Mental retardation, Aphasia, Shuffling gait, and Adducted thumbs) syndrome, X-linked complicated spastic paraplegia type I (SPG1) and X-linked Agenesis of the Corpus Callosum (ACC). These four disorders represent a clinical spectrum that varies both between and within families. The main clinical features of this spectrum are Corpus callosum hypoplasia, mental Retardation, Adducted thumbs, Spastic paraplegia and Hydrocephalus (CRASH syndrome). Since there is no biochemically assayed disease marker, molecular analysis of the L1CAM gene is the only means of confirming a clinical diagnosis. Most L1CAM mutations reported to date are point mutations (missense, nonsense, splice site) and only a few patients with larger rearrangements have been documented. We have characterised a rare intragenic deletion of the L1CAM gene in a sample of DNA extracted from a chorionic villus biopsy (CVB) performed at 12 weeks' gestation. =, (Copyright 2005 John Wiley & Sons, Ltd.)

Pseudoachondroplasia (PSACH) and some forms of multiple epiphyseal dysplasia (MED) result from mutations in the gene encoding cartilage oligomeric matrix protein (COMP). COMP is a large pentameric glycoprotein found predominantly in the extracellular matrix of cartilage, tendon, and ligament. As a modular protein, it is composed of a coiled-coil domain, four type II (T2) repeats, eight type III (T3) repeats, and a large globular C-terminal domain (CTD). The majority (>85%) of COMP mutations causing PSACH or MED are found in the exons encoding the T3 repeats, and the disease mechanism has been characterised in detail. Much less is known about disease-causing mutations in the CTD; in 10 years only seven mutations have been identified. In this study, we describe eight novel and two recurrent mutations that we have recently identified in patients with PSACH or MED. Interestingly, these mutations result in a spectrum of disease, ranging from mild MED to severe PSACH. Mapping of all known COMP CTD mutations on a three-dimensional model of the C-terminal domain shows that the CTD mutations cluster in two distinct regions. These regions are probably important in stabilising the T3-CTD structure and mediating intra- or intermolecular interactions.

The skeletal dysplasias are a clinically and genetically heterogeneous group of conditions affecting the development of the osseous skeleton and fall into the category of rare genetic diseases in which the diagnosis can be difficult for the nonexpert. Two such diseases are pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), which result in varying degrees of short stature, joint pain and stiffness and often resulting in early onset osteoarthritis. PSACH and some forms of MED result from mutations in the cartilage oligomeric matrix protein (COMP) gene and to aid the clinical diagnosis and counselling of patients with a suspected diagnosis of PSACH or MED, we developed an efficient and accurate molecular diagnostic service for the COMP gene. In a 36-month period, 100 families were screened for a mutation in COMP and we identified disease-causing mutations in 78% of PSACH families and 36% of MED families. Furthermore, in several of these families, the identification of a disease-causing mutation provided information that was immediately used to direct reproductive decision-making.

We describe the development and implementation of a neurofibromatosis type 2 (NF2) mutation scanning service based on novel techniques. All 17 exons of the NF2 gene are amplified in four polymerase chain reaction (PCR) reactions, using the meta-PCR technique to link the NF2 exons into chimeric concatamers. The meta-PCR products are then scanned for point mutations by direct sequencing. A four-exon dosage assay is used to test for large deletion/duplication mutations. In certain cases when tumour studies are necessary, these techniques are also combined with loss of heterozygosity analysis with three highly polymorphic microsatellite markers located within or close to the NF2 gene. Over a period of 2 years, we have applied these techniques in a service setting to the analysis of 271 patient samples (245 lymphocyte DNA; 26 schwannoma DNA). Meta-PCR and sequencing identified 90 point mutations in the 271 blood and tumor samples, 48 of which have not been reported previously. Dosage analysis identified large deletions in 12 of the lymphocyte DNA samples. In addition, over 84% of mutations were identified in 23 schwannoma DNA samples in which complete analysis was possible. Adoption of this novel strategy has increased the overall mutation detection rate in familial NF2 cases to 88% and sporadic NF2 cases to 59%. It has also allowed us to decrease our reporting turnaround times, and because of a low overall failure rate, permitted the running of an efficient and cost-effective service.

The safe application of new strategies for the treatment of graft-versus-host disease (GVHD) is hampered by the lack of a clinically relevant model for preclinical testing. Current models are based on intraperitoneal transfer of human peripheral blood mononuclear cells (huPBMCs) into NOD-SCID (nonobese diabetic-severe combined immunodeficient)/SCID mice. Intravenous transfer would be preferred but this has always been ineffective. We developed a new model for xenogeneic GVHD (X-GVHD) by intravenous transfer of huPBMCs into RAG2-/- gammac-/-mice. Our results show a high human T-cell chimerism of more than 20% (up to 98%) in more than 90% of mice, associated with a consistent development of XGVHD within 14 to 28 days and a total mortality rate of 85% shorter than 2 months. After murine macrophage depletion, engraftment was earlier and equally high with lower doses of huPBMCs. Human macrophages were also absent in these mice. Purified huCD3+ cells showed a similar X-GVH effect with contribution of both CD4 and CD8 phenotypes. Human immunoglobulins and cytokines were produced in diseased mice. One of 30 mice developed chronic X-GVHD with skin histology similar to human GVHD. In conclusion, we present a new model for X-GVHD by intravenous transfer of huPBMCs in RAG2-/- gammac-/- mice. Murine and human macrophages do not seem to be necessary for acute X-GVHD in this model.

Allogeneic donor T lymphocytes manipulated genetically to express the herpes simplex virus thymidine kinase (HSV-TK) gene have emerged as promising tools to alter the balance between graft versus host disease and graft versus leukemia after allogeneic stem cell transplantation, since they can be eliminated selectively in vivo with ganciclovir. Recently, it was reported that in SFCMM-3, an HSV-TK-encoding retroviral vector, two cryptic splice sites in the HSV-TK sequence led to the generation of an HSV-TK splice variant (deltaHSV-TK) that encodes a ganciclovir-resistant gene product. In order to quantify wtHSV-TK and deltaHSV-TK RNA levels we have developed two real time Taqman PCR assays. We demonstrate that the sensitivity of both PCR assays is 10(-4). It was found that the splice variant is generated in the packaging cell line and results in approximately 4.8+/-1.9% of virions that contain deltaHSV-TK RNA. After transduction of human T cells no significant increase in deltaHSV-TK RNA could be detected. Thus, at maximum 4.2+/-1.2% of T cells transduced with SFCMM-3 will be resistant to ganciclovir due to this mechanism only. Together, these assays provide a powerful method to monitor patients in future clinical trials.

This paper reports a European pilot External Quality Assessment (EQA) scheme for the molecular diagnosis of Huntington's disease (HD). The European Molecular Genetics Quality Network (EMQN) chose HD as a relatively simple assay that allows a range of difficulty in terms of technical competence and interpretation. Fourteen centres from 12 different countries participated. The scheme organiser provided five cases together with mock clinical information. The participating laboratories were asked to complete the analyses and return the reports in English to their normal laboratory format within a fixed period. The scheme demonstrates a level of potential misdiagnosis in molecular analysis of HD as well as a wide variety in way of reporting laboratory results. Overall 9/146 (6.2%) of alleles fell outside the set limits, and the rate of misdiagnosis was 1/78 (1.3%). A closer estimate of diagnostic accuracy will require expansion of the scheme.

Many mutation scanning techniques are capable of locating mutations in DNA fragments much larger than the average exon. We have developed a system called Meta-PCR that can maximize the length of sequence scanned by these techniques, improving their productivity and realizing their full potential. Meta-PCR is a simple, versatile, and powerful method for generating chimeric DNA molecules. Currently, up to five PCR amplifiable fragments can be combined to form a single linear amplimer. The Meta-PCR reaction is self-assembling and takes place in two coupled stages carried out in a single reaction vessel. The order of fragments is reproducible and determined by primer design. We have developed two Meta-PCR assays, one comprising exons 6-10 of the Neurofibromatosis type 2 (NF2) gene and the second exons 8-12 of the human mismatch repair gene, hMLH1. We verified by direct sequencing that the order and sequence of the component exons in the Meta-PCR products is as predicted. Meta-PCR products from seven previously ascertained heterozygotes for NF2 mutations were directly sequenced. All seven mutations were clearly visible as mixed bases at the expected nucleotide, confirming that Meta-PCR faithfully reproduces the original sample genotype. We have evaluated the downstream use of the NF2 Meta-PCR products in fluorescent solid-phase chemical cleavage of mismatches (CCM). Meta-PCR products from eleven NF2 mutant heterozygotes were screened retrospectively for piperidine cleavage after hydroxylamine or potassium permanganate modification of mismatched bases. Ten of the 11 mutants were detected by visible cleavage. One mutation predicted to be cleaved after potassium permanganate modification was not detected. However, we were able to attribute this false negative to a failure in the CCM method. Meta-PCR is likely to be useful to clinical molecular diagnostic laboratories, helping them to fulfill demand for rapid and accurate screening for point mutations in large multi-exon genes.

Objectives: In view of the recent reports of recurrent mutations in BRCA1 and BRCA2 in the Ashkenazi Jewish population, we have undertaken to assess the frequency of these mutations in this population attending for genetic counselling and risk assessment of familial breast cancer., Design: Mutation screening for the 185delAG and the 5382insC mutations in BRCA1 and the 6174delT mutation in BRCA2 was performed on DNA samples from either subjects affected by breast or ovarian cancer or obligate gene carriers. The likelihood of the cancers being hereditary in each family was calculated., Subjects: Blood samples were obtained from 26 affected subjects or obligate gene carriers from 23 Ashkenazi Jewish families, all with a history of either early onset breast or ovarian cancers, or multiple cases of breast or ovarian cancer., Results: Twelve mutations have been identified in the 23 families (52%) of which eight (67%) were the 185delAG mutation, three (25%) were the 6174delT mutation, and one (8%) was the 5382insC mutation. While the majority of these mutations were identified in families with a greater than 50% probability of being hereditary under the CASH segregation model, three mutations were identified in families with a 35% or less probability., Conclusions: Genetic screening of the recurrent mutations in Ashkenazi Jewish families will lead to the availability of predictive testing in a reasonably large proportion, even if the family history of breast/ovarian cancer is not particularly strong. In our view it is possible to reassure high risk unaffected members of these families, if the screening is negative for these mutations, even if a sample from an affected member of the family is unavailable for previous screening.

Clinical molecular genetics has recently become recognized as a diagnostic discipline. This article covers the evolution, structure, and possible forward development of clinical molecular genetics. Topics covered include general test categories, introducing new tests, laboratory facilities, staffing and training, and overview of quality issues.

Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.

In the present study, leukocyte DNA from 143 patients with familial clustering of breast and/or ovarian cancer and tumour DNA from 96 breast carcinomas were screened for base mutations in the estrogen receptor gene (ESR). Three patients with a family history of cancer were carrying a Gly160Cys germline substitution. This alteration was also detected in eight (four females and four males) of 729 controls (366 female, 363 males), indicating that the substitution probably represents a polymorphism. However, in the 229 female controls in whom family history of cancer was known, one of two who had a sister with breast cancer was carrying the variant allele. Hence, a possible clinical significance of the glycine into cysteine cannot be completely ruled out and should be further investigated. Somatic mutations were not detected in any of the tumours studied, and the present data do not provide support for somatic ESR base mutations as an important mechanism for hormonal therapy resistance in estrogen receptor-positive breast carcinomas.

Clinical molecular genetics has only recently become recognizable as a diagnostic discipline in its own right-gradually becoming distinct from its academic-and research-based origins. This chapter seeks to give some shape and context to the contrtbutions that follow and add to previously published ideas of how diagnostic laboratories are structured and evolving (1, 2). The chapter largely draws on the UK experience of the field and does not claim to be authoritative on developments in North America, Europe, Australasia, or other parts of the world.

Diagnosis of autosomal dominant adult polycystic kidney disease (APKD) is possible by ultrasonographic scanning (USS) or by using DNA markers linked to the PKD1 locus. Ultrasonography is complicated by the age dependent penetrance of the gene and linkage studies are subject to recombination errors owing to meiotic crossing over and locus heterogeneity. This study draws on data collected from a voluntary family register of APKD over 10 years. Records of 150 families were examined, ultrasound reports were obtained from 242 people at 50% prior risk, and 37 families were typed for DNA markers. The fraction of APKD resulting from loci unlinked to PKD1 (designated PKD2 here) was calculated at 2.94% (upper confidence limit 8.62%). Some subjects who were negative on initial scan later gave a positive scan, but there was no example of a definite gene carrier aged over 30 giving a negative scan. In families large enough for linkage analysis, most people who were at 50% prior risk could be given a final risk below 5% or above 95%, by using combined ultrasound and DNA studies.

Twenty-four families with adult onset polycystic kidney disease were typed for markers flanking the PKD1 locus on chromosome 16. The aggregated results gave a significant lod score in favour of linkage to PKD1. Within this group of families two showed unusual features: recombinations, including double recombinations, and, in one family, an unexpectedly high proportion of affected people. We consider the evidence that in these families the disease might result from a mutation at a different locus, PKD2, not linked to PKD1. We suggest that a useful test is to compare the relative numbers of meioses apparently non-recombinant and doubly recombinant for markers flanking the normal disease locus, ignoring meioses recombinant for only a single marker. Using this test, neither our two families nor the data published so far on other families provide compelling evidence for the existence of a second locus for adult polycystic kidney disease. For genetic counselling in families too small to give internal evidence for or against linkage, the extra uncertainty can be handled by using a higher recombination rate.

96 families at risk of having a child with cystic fibrosis have been counselled about prenatal diagnosis by the use of linked DNA probes. Of the first 30 pregnancies 9 children have been born and confirmed to be free from CF, as predicted; 8 pregnancies were terminated (1 of these was found to be miscarrying at time of termination); 12 pregnancies, in which the fetuses are predicted to be unaffected, are in progress; and 1 has miscarried. No couple with a prenatal prediction of CF decided to continue with the pregnancy. 1 pregnancy was terminated because of a 50:50 chance of an affected fetus. In 1 twin pregnancy the affected fetus was selectively aborted, and the other baby was confirmed after birth to be unaffected, as predicted. In the other twin pregnancy the two chorionic plates could not be samples separately; the pregnancy was continued on the basis of normal findings for the lower plate, and both twins have turned out to be normal. Many couples at risk see early prenatal diagnosis as a way to help them have an unaffected child.

Seventy families with Duchenne muscular dystrophy (DMD) known to the Institute of Child Health fall into three categories with respect to potential linkage analysis with the X chromosome DNA markers RC8 and L1.28 that bridge the DMD gene. Families in which there is at least one obligatory female heterozygote (n = 13). Here 'prediction' and 'exclusion' of DMD gene transmission may be possible, the accuracy being dependent on the closeness of the linkage of the DNA marker(s) to the DMD gene; an illustrative case is reported. Families in which there is a single affected boy, who also has one or more healthy brothers (n = 26). Given an informative restriction fragment length polymorphism (RFLP), the probability that the boy represents a new mutation can be reassessed; it is also possible to 'exclude' the DMD gene in a sister. Families with a single affected boy with no brother (n = 30). Here 'exclusion' of the DMD gene in a sister may be possible. Only in one family was there no possibility of useful linkage analysis. The linkage analysis required is described, and the need to check DMD families for informative RFLPs is stressed.

A library of 50,000 recombinants representative of the human X chromosome has been constructed. Human X chromosomes were physically separated using a fluorescence-activated cell sorter. The DNA was purified from the chromosomes, digested to completion with the restriction enzyme EcoRI and cloned into the phage lambda gtWES.lambda B. The X-derived nature of the recombinants was confirmed by hybridization to rodent/human cell line DNA containing only the human X chromosome. Such libraries will be particularly useful for the investigation of genetic diseases such as Duchenne muscular dystrophy, where the basic defect has not been elucidated, and of neoplasia, where several specific chromosomal anomalies, particularly for the leukaemias, have been identified.

A particular haplotype defined by probes XV2c, KM19, and CS.7 at the D7S23 locus was found on 90% of chromosomes which carry cystic fibrosis (CF), but on only 11% of normal chromosomes in a UK sample of CF carriers. We show how such data can be used to calculate carrier risks for people with and without a family history of CF, and give examples of clinical applications. For parents or sibs of dead CF patients, phase and genotypes can often be assigned with only 1 to 2% error. However, this method is not suitable for prenatal testing where there is no history of CF; for couples with no family history, no fetus can be shown to be at more than 2% risk of being affected.

The expression of HLA class I alpha chains by clones derived from a human X mouse T-cell line containing human chromosomes 6 and 15 was analysed by immunofluorescence. Evidence is presented showing that the expression of HLA class I alpha chains is associated with HLA-beta 2m. This is based on the phenotypic and quantitative fluorescence flow cytometric analysis of a series of cloned hybrids. In contrast, HLA and H-2 class I determinants show no evidence of such correlation. Results of cell-sorting indicate that the loss of HLA class I alpha chain expression is due to phenotypic modulation, in addition to gene segregation.

Chorionic villi can be obtained by direct transcervical aspiration at 9 to 10 weeks' gestation and used for analysis of fetal DNA. However, for the method to be reliable, there must be no detectable contamination by maternal DNA. To investigate the question of contamination, we compared the DNA of chorionic villi from five fetuses with that obtained from maternal lymphocytes, using the restriction endonuclease Taql and specific DNA probes for a pair of alleles on the X chromosome. The alleles yield fragments of different lengths when digested with Taql (length polymorphism), which can be demonstrated by electrophoresis and hybridization with the radioactive DNA probes. If the pattern obtained with the chorionic DNA is different from that obtained with the maternal DNA, contamination is not present. In two cases the fetal DNA of the chorionic villi was shown to be uncontaminated by maternal tissue. In one of these cases the mother was heterozygous and the fetus was homozygous; in the other the mother was homozygous and the fetus was heterozygous. In three other cases no definitive conclusions could be drawn, because the genotypes of the fetus and mother were identical. We conclude that chorionic villi at 9 to 10 weeks' gestation are a source of fetal DNA that can be used for gene analysis, with no detectable contamination by maternal DNA.

A total of 102 families with Duchenne muscular dystrophy has been studied with linked DNA polymorphisms as an aid to estimating carrier risks for female relatives. Early work using probes RC8, L1.28, and pXUT23 gave very little clinically useful information because of the high recombination rates between these probes and Duchenne muscular dystrophy and the low proportion of women who were heterozygous. Clinically useful results were obtained using probes 99-6, 754, and particularly pERT87. Examples are given of deductions which can be made using these probes. The importance of a genetic register is stressed as a tool for long term contact with the families and other professionals.

A recent report from the Departments of Health draws attention to the value of DNA diagnosis for inherited diseases and the need for planning these services in the National Health Service. There is great potential for preventive medicine, but a major immediate benefit is the newfound ability to exclude the carrier state in many people at risk and to protect fetuses from abortion when, as in most cases, they are shown to be normal by DNA tests. However, the widespread application of these new techniques requires prior evaluation and general acceptance. This will only be obtained after public debate, education of professionals and the population, and the establishment of adequate non-directive genetic counselling services. Some of the points to be considered in setting up molecular genetics laboratories are described.